Compositions and Methods for Treatment of Myotonic Dystrophy Type 1 with CRISPR/SluCas9

Abstract

Compositions and methods for treating Myotonic Dystrophy Type 1 (DM1) are encompassed.

1 . A composition comprising a single nucleic acid molecule encoding one or more guide RNAs and a Cas9, wherein the single nucleic acid molecule comprises:
 a. a first nucleic acid encoding one or more spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531 and a second nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9);   b. a first nucleic acid encoding one or more spacer sequence comprising at least 20, or 21 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531 and a second nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9);   c. a first nucleic acid encoding one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1-65, 67-167, and 201-531 and a second nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9);   d. a first nucleic acid encoding 2 spacer sequences selected from any one of SEQ ID NOs: 63 and 100, and 64 and 100, and a second nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9); or   e. a first nucleic acid encoding one or more spacer sequence selected from any one of SEQ ID NOs: 8, 63, 64, and 81 and a second nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9).   
     
     
         2 . The composition of  claim 1 , further comprising a DNA-PK inhibitor. 
     
     
         3 . The composition of  claim 2 , wherein the DNA-PK inhibitor is Compound 6, Compound 1, or Compound 2. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The composition of  claim 1 , wherein the guide RNA is an sgRNA. 
     
     
         7 . The composition of  claim 1 , wherein the guide RNA is a modified guide RNA. 
     
     
         8 .- 11 . (canceled) 
     
     
         12 . The composition of  claim 1 , wherein the single nucleic acid molecule is associated with a lipid nanoparticle (LNP), or wherein the single nucleic acid molecule is associated with a viral vector. 
     
     
         13 . (canceled) 
     
     
         14 . The composition of  claim 12 , wherein the single nucleic acid molecule is associated with a viral vector, and wherein the viral vector is an adeno-associated virus vector, a lentiviral vector, an integrase-deficient lentiviral vector, an adenoviral vector, a vaccinia viral vector, an alphaviral vector, or a herpes simplex viral vector. 
     
     
         15 .- 22 . (canceled) 
     
     
         23 . The composition of  claim 1 , comprising a nucleic acid encoding SluCas9, wherein the SluCas9 comprises the amino acid sequence of any one of SEQ ID NOs: 712 or 718-720. 
     
     
         24 .- 31 . (canceled) 
     
     
         32 . A method of treating Myotonic Dystrophy Type 1 (DM1), the method comprising delivering to a cell a single nucleic acid molecule comprising a nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9); and:
 i) a nucleic acid encoding a guide RNA, wherein the guide RNA comprises:
 a. one or more spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; 
 b. one or more spacer sequence comprising at least 20, or 21 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or 
 c. one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or 
   ii) a nucleic acid encoding a pair of guide RNAs comprising:
 a. a first spacer sequence selected from any one of SEQ ID NOs: 1-65 and a second spacer sequence selected from any one of SEQ ID NOs: 67-167; 
 b. a first and second spacer sequence comprising at least 20, or 21 contiguous nucleotides of any of the first and second spacer sequences of i) a.; 
 c. a first and second spacer sequence that is at least 90% identical to any of the first and second spacer sequences of i) a. or i) b.; 
 d. a first and second spacer, or one or more vectors encoding the pair of guide RNAs, wherein the first and second spacer sequences comprise any one of the following pairs of SEQ ID NOs; 6 and 72; 6 and 81; 6 and 84; 6 and 98; 6 and 100; 6 and 114; 6 and 122; 6 and 134; 6 and 139; 6 and 149; 6 and 166; 8 and 72; 8 and 72; 8 and 81; 8 and 84; 8 and 98; 8 and 100; 8 and 114; 8 and 122; 8 and 134; 8 and 139; 8 and 149; 8 and 166; 10 and 72; 10 and 81; 10 and 84; 10 and 98; 10 and 100; 10 and 114; 10 and 122; 10 and 134; 10 and 139; 10 and 149; 10 and 166; 21 and 72; 21 and 81; 21 and 84; 21 and 98; 21 and 100; 21 and 114; 21 and 122; 21 and 134; 21 and 139; 21 and 149; 21 and 166; 58 and 72; 58 and 81; 58 and 84; 58 and 98; 58 and 100; 58 and 114; 58 and 122; 58 and 134; 58 and 139; 58 and 149; 58 and 166; 62 and 72; 62 and 81; 62 and 84; 62 and 98; 62 and 100; 62 and 114; 62 and 122; 62 and 134; 62 and 139; 62 and 149; 62 and 166; 63 and 72; 63 and 81; 63 and 84; 63 and 98; 63 and 100; 63 and 114; 63 and 122; 63 and 134; 63 and 139; 63 and 149; 63 and 166; 64 and 72; 64 and 81; 64 and 84; 64 and 98; 64 and 100; 64 and 114; 64 and 122; 64 and 134; 64 and 139; 64 and 149; and 64 and 166; or 
 e. a first and second spacer, or one or more vectors encoding the pair of guide RNAs, wherein the first and second spacer sequences comprise SEQ ID NOs: 63 and 100 or SEQ ID NOs: 64 and 100. 
   
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . A method of excising a CTG repeat in the 3′ UTR of the DMPK gene, the method comprising delivering to a cell a single nucleic acid molecule comprising a nucleic acid encoding a  Staphylococcus lugdunensis  Cas9 (SluCas9); and:
 i) a nucleic acid encoding a guide RNA, wherein the guide RNA comprises:
 a. one or more spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; 
 b. one or more spacer sequence selected from any one of SEQ ID NOs: 8, 63, 64, and 81; 
 c. one or more spacer sequence comprising at least 20, or 21 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or 
 d. one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or 
 
 ii) a nucleic acid encoding a pair of guide RNAs comprising:
 a. a first spacer sequence selected from any one of SEQ ID NOs: 1-65 and a second spacer sequence selected from any one of SEQ ID NOs: 67-167; 
 b. a first and second spacer sequence comprising at least 20, or 21 contiguous nucleotides of any of the first and second spacer sequences of i) and; 
 c. a first and second spacer sequence that is at least 90% identical to any of the first and second spacer sequences of i) a. or i) b.; or 
 d. a first and second spacer sequence selected from any one of SEQ ID NOs: 63 and 100, and SEQ ID NOs: 64 and 100. 
 
 
     
     
         36 . (canceled) 
     
     
         37 . (canceled) 
     
     
         38 . The method of  claim 32 , comprising administering a DNA-PK inhibitor. 
     
     
         39 . The method of  claim 38 , wherein the DNA-PK inhibitor is Compound 6, Compound 1, or Compound 2. 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . The method of  claim 32 , wherein the SluCas9 comprises the amino acid sequence of any one of SEQ ID NOs: 712 or 718-720. 
     
     
         43 . (canceled) 
     
     
         44 . (canceled) 
     
     
         45 . The composition of  claim 1 , wherein the nucleic acid encoding the gRNA or the nucleic acid encoding the pair of gRNAs comprises a sequence selected from any one of SEQ ID NOs: 600-601, or 900-917. 
     
     
         46 . (canceled) 
     
     
         47 . The composition of  claim 1 , wherein the nucleic acid molecule encodes at least a first guide RNA and a second guide RNA, and wherein the nucleic acid molecule further encodes a spacer sequence for the first guide RNA, a scaffold sequence for the first guide RNA, a spacer sequence for the second RNA, and a scaffold sequence for the second guide RNA. 
     
     
         48 .- 52 . (canceled) 
     
     
         53 . The composition of claim  48 , wherein the scaffold sequence for the first guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 901-916, and wherein the scaffold sequence for the second guide RNA comprises a different sequence selected from the group consisting of SEQ ID NOs: 901-916. 
     
     
         54 .- 61 . (canceled) 
     
     
         62 . A composition comprising a first nucleic acid molecule and a second nucleic acid molecule, wherein the first nucleic acid molecule encodes a  Staphylococcus lugdunensis  Cas9 (SluCas9) and the second nucleic acid molecule encodes one or more guide RNAs comprising:
 a. one or more spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531;   b. one or more spacer sequence comprising at least 20, or 21 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or   c. one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1-65, 67-167, and 201-531.   
     
     
         63 . The composition of  claim 62 , wherein the first nucleic acid molecule does not encode a guide RNA. 
     
     
         64 . The composition of  claim 62 , wherein the first nucleic acid molecule encodes:
 a. one or more spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531;   b. one or more spacer sequence comprising at least 20, or 21 contiguous nucleotides of a spacer sequence selected from any one of SEQ ID NOs: 1-65, 67-167, and 201-531; or   c. one or more spacer sequence that is at least 90% identical to any one of SEQ ID NOs: 1-65, 67-167, and 201-531.   
     
     
         65 .- 87 . (canceled)